Summary of Work: The study of oxidative stress has attracted considerable interest and has been the focus of much research in recent years. Cumulative oxidative damage to tissues has been implicated in a number of disease states, e.g. the aging process, cancer, and ischemia reperfusion. The study of oxidative stress in the mitochondria has shown that hydrogen peroxide is produced via the incomplete reduction of oxygen during oxidative phosphorylation. Hydrogen peroxide levels are kept relatively low under normal physiological conditions. Under certain conditions, such as inflammation, excessive amounts of hydrogen peroxide are produced. The production of excess hydrogen peroxide is thought to precede several occurrences, such as lipid peroxidation, DNA and/or protein damage, and glutathione depletion, that are characteristic of oxidative stress. We have probed the role of cytochrome C free-radicals in oxidative stress by studying the anaerobic reaction between horse heart cytochrome C (HHCC) and hydrogen peroxide using the spin-trapping reagent 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS). The MS of the reaction product clearly showed the presence of at least four distinct DBNBS adducts. Enzymatic digest followed by LC/ESI/MS showed an ion due to the DBNBS adduct with Tyr74 of the HHCC which is on the surface of the protein. It is thought that the heme is initially oxidized and then the radical site is transferred intramolecularly to the tyrosine. We are currently identifying other sites of radical formation based on the MS/MS analysis of the proteolytic digest of the DBNBS spin-trapped protein. In parallel experiments we are also investigating the products using MNP as a spin-trap. Investigation of cyanyl radical adducts of cytochrome c oxidase. Cyanide is a frequently used inhibitor of mitochondrial respiration. It has been widely accepted that cyanide functions as a bridge between Heme a3 and CuB. We have observed using ESR that cyanide can be oxidized to the cyanyl radical by cytochrome c oxidase [3]. It is possible that this radical can act as a suicide inhibitor by undergoing reaction with the heme to form a covalent adduct. We have investigated this reaction, and have observed that cyanyl radical does form a covalent adduct with heme. MS/MS showed that the cyanide is attached to the porphyrin ring.